From our collaborative publications this year, we report the following advances: We are interested in developing animal models that can better predict the efficacy of malaria vaccines being considered for human trials. In this study led by colleagues at the FDA (Chawla B, et al, Infection and Immunity) we contributed recombinant CSP from Plasmodium yoelli (rPyCSP), which when delivered in Montanide ISA 51 adjuvant, induced sterilizing immunity against sporozoite challenge in C57BL/6 and BALB/c strains of mice. Protection against sporozoite challenge correlated with the higher frequency of CD4+ T cells that express IL-2, IL-4 and TNF-. In human studies of the RTS,S vaccine (a CSP-based vaccine), clinical immunity was associated with higher IgG levels and greater frequency of IL-2 and TNF- producing CD4+ T cells. These results provide an excellent model system to evaluate the efficacy of novel adjuvants, vaccine dosage and determine the correlates of immunity in search for superior candidate malaria vaccines. 18S rRNA is a biomarker that provides an alternative to thick blood smears in controlled human malaria infection (CHMI) trials. Together with colleagues at multiple institutions led from the University of Washington (Seilie A, et al. AJTMH), we validated a multiplex quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for Plasmodium 18S rRNA, prospectively compared blood smears and qRT-PCR for three trials, and modeled treatment effects at different biomarker-defined parasite densities to assess the impact on infection detection, symptom reduction, and measured intervention efficacy. We determined that Plasmodium 18S rRNA is a sensitive and specific biomarker that can justifiably replace blood smears for infection detection in CHMI trials in non-endemic settings. This study led to biomarker qualification through the U.S. Food and Drug Administration for use in CHMI studies at non-endemic sites, which will facilitate biomarker use for the qualified context of use in drug and vaccine trials. Our unpublished progress during this reporting period includes the following advances: Sterile protection against malaria infection can be induced by multiple exposures to radiation-attenuated sporozoite (RAS) parasite forms in mice and humans, if the RAS remain sufficiently viable to invade hepatocytes. Our clinical trials of PfSPZ-based RAS vaccines have consistently shown that antibody responses to CSP are associated with protection of naturally acquired P. falciparum infection. Thus, new subunit vaccine strategies are needed. CSP tolerant transgenic mice are also protected after RAS immunization, implicating additional pre- erythrocytic antigens as targets of sterile immunity. RTS,S based on Pf circumsporozoite protein (PfCSP), the predominant sporozoite surface antigen, is the most advanced subunit candidate, but has shown only partial efficacy against malaria episodes in Phase III testing. We have sought to identify novel candidate pre-erythrocytic vaccine antigens (PEVA) that could add to the level of protection achieved with CSP immunogens alone. We assume that PEVA candidates are transcribed during liver stage (LS) development and have used transcriptomic data developed in our lab to identify such candidate antigens. LMIV has assessed the protective efficacy of some of these immunogens by DNA vaccination in rodent models of malaria. In FY2019, we down-selected to 2 PyPEVA candidates to pursue the PfPEVA orthologues. The two candidates selected were SHMT and 305w. We also pursued expression of both PyPEVA and PfPEVA in E. coli instead of insect cells. Stability of the protein was increased by modifying the N-terminal amino acid to increase the half-life of the protein in E. coli and as a result, protein expression was observed for PySHMT, PfSHMT and Pf305w. Some initial refolding conditions to achieve soluble protein have been identified. In FY2019, we developed new reagents to test strain-specific and domain-specific reactivity of PfCSP from patients in the whole organism PfSPZ vaccine trial. Peptides were designed from different domains in PfCSP that had evidence of sieving. C-terminal peptides of CSP (TSR) that form structure were generated recombinantly as a SUMO fusion with a site-specific biotin tag for specific attachment to Bio-Plex beads. We generated milligram quantities of cleaved and biotinylated peptides. We tested these peptides for correct structure by testing reactivity to a mouse monoclonal antibody that reacts to folded TSR structure and has activity. First, we determined that the NF54 TSR peptide recognizes the peptide, suggesting our peptide is correctly folded. Second, we observed that strain-specific peptides reduced binding to the monoclonal antibody.